Acinetobacter lysins

ABSTRACT

Acinetobacter  lysin polypeptides and variants peptides with killing activity against gram negative bacteria. Methods for treating bacterial infections or bacterial colonization using  Acinetobacter  lysin polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a United States National Phase of PCT/US2015/037962,filed Jun. 26, 2015, which claims the benefit of priority to U.S.Provisional Application Ser. No. 62/017,618, filed Jun. 26, 2014. Theentire contents of which are incorporated herein by reference.

SEQUENCE LISTING

This application incorporates by reference in its entirety the sequencelisting entitled “235932-406445_Sequence_Listing_ST25”, (57 KB) whichwas created on Jan. 18, 2017, and filed electronically herewith.

FIELD

Compositions comprising a bacteriophage lytic enzyme specific forAcinetobacter and method for treating Acinetobacter infections.

BACKGROUND

Acinetobacter baumannii-calcoaceticus complex and other members of thisspecies frequently colonize the human skin without harm. However,injuries to the skin from scrapes, wounds or surgery, can result inAcinetobacter infection of the wound, blood, soft tissues, and centralnervous system. Given that >80% of Acinetobacter sp. are also multiplydrug resistant (MDR) (at least three classes of antibiotics), theseinfections may result in adverse clinical outcomes, including high ratesof morbidity and mortality, prolonged hospital stay, and substantialhealth care expenses. Military personnel and athletes have an increasedthe risk of injuries (from skin abrasions to severe wounds) that wouldbe susceptible to infection by Acinetobacter spp., thus methods toremove them quickly and effectively would reduce or eliminate downstreamcomplications. Outbreaks caused by MDR Acinetobacter have been reportedin hospitals all over the world; more recently, they have become aserious problem in military medical facilities. Because of its MDR,Acinetobacter infections are difficult to treat so infections by theseorganisms usually result in a poor outcome. Thus, new and better ways ofcontrolling this pathogen are needed.

Acinetobacter baumannii strains resistant to all known antibiotics havenow been reported. Acting in synergy with this emerging resistanceprofile is the uncanny ability of A. baumannii to survive for prolongedperiods throughout a hospital environment, thus potentiating its abilityfor nosocomial spread. The organism commonly targets hospitalizedsubjects, who are critically ill with breaches in skin integrity andairway protection. As such, hospital-acquired pneumonia is still themost common infection caused by A. baumannii. However, recently,infections involving the central nervous system, skin and soft tissue,and bone have emerged as highly problematic for certain institutions.Because of this resistance problem, new methods to control thesepathogens must be developed.

Antimicrobial agents known as bacteriophage-encoded lysins have beenidentified. Bacteriophages are viruses that infect bacterial and it isestimated that there are 10⁶ distinct bacteriophage species.Bacteriophage lysins are generally genus- or species-specific, i.e., aStaphylococcus aureus phage lysin may have activity only againstStaphylococcus aureus providing a targeted therapeutic approach. In somecases, lysins may have activity against several genera or species.

Bacteriophage infect their host bacteria to produce more virusparticles. At the end of the reproductive cycle they are faced with aproblem, how to release the progeny phage trapped within the bacterium.They solve this problem by producing an enzyme called “lysin” thatdegrades the cell wall of the infected bacteria to release the progenyphage. The lytic system consists of a holin and at least onepeptidoglycan hydrolase, or lysin, capable of degrading the bacterialcell wall. Typically, the holin is expressed in the late stages of phageinfection forming a pore in the cell membrane, allowing the lysin(s) togain access to the cell wall peptidoglycan resulting in release ofprogeny phage. Significantly, exogenously added lysin, in the absence ofa holin, can lyse the cell wall of healthy, uninfected cells, producinga phenomenon known as “lysis from without”.

SUMMARY

We have recently identified, purified and characterized several phagelysins that specifically attack Acinetobacter bacteria. This is abreakthrough since most lysins have antibacterial activity only againstgram-positive bacteria. The purified phage lysins of the presentinvention are well suited for a variety of applications such astreatment of bacterial infections, and disinfection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Negative staining electron micrograph showing phage inducedfrom A. baumannii strain 1790.

FIG. 1B. Negative staining electron micrograph showing phage inducedfrom A. baumannii strain 1794

FIG. 1C. Negative staining electron micrograph showing phage inducedfrom A. baumannii strain 1796

FIG. 2. A representative image of lysin clone activity in clearing liveA. baumannii imbedded in the agar.

FIG. 3. Schematic of amino acid sequences of cloned lysins showing fourclasses of lytic activity: i) glycosyl hydrolase family, ii) phagebaseplate lysozymes, iii) lysozyme autolysins, and iv) lysins.

FIG. 4. Alignment of nucleotide sequences for cloned lysins.

FIG. 5. Alignment of amino acid sequences of cloned lysins.

FIG. 6. Is a graph showing the lytic activity of 21 cloned constructsagainst thirteen. different A. baumannii clinical isolates.

FIGS. 7A, and 7B. Blebbing of the cytoplasmic membrane containingcytosolic contents from A. baumannii cells are observed after treatmentwith F307 (arrows).

FIG. 8. Scanning electron micrograph of 3-day biofilms of strain 1791 A.baumannii before and after treatment with F307 polypeptide.

FIG. 9. Is a graph showing reduction in bacterial counts on wholecatheter pieces with Acinetobacter biofilm after treatment with F307polypeptide.

FIG. 10. Is a graph showing the survival of mice infected with A.baumannii treated with F307 polypeptide versus control.

FIG. 11. Sequence of F307, P307 polypeptide without and with shortextension (P307Ex).

FIG. 12. FIG. 12A is a graph a comparison of in vitro bactericidalactivities of P307, P307SQ-8C and P307_(AE-8) against A. baumanniistrains #1791, S5 and ATCC17978. FIG. 12B shows the comparative in vivobactericidal activity of P307, P307SQ-8C, and P307CS-8 against A.baumannii strains #1791 and S5. FIG. 12C shows a comparison of thecomparative in vivo bactericidal activity of P307SQ-8C and P307CS-8against A. baumannii strains #1791, S5 and ATCC17978.

FIG. 13. The in vitro bactericidal activities of P307 and P307SQ-8Cagainst A. baumannii strain #1791 to investigate the pH optimum (13A),and NaCl optimum (13B). The same conditions, except for the variables,were used with 50 mM Tris-HCl, pH 7.5 to determine the concentrationoptimum (13C), and killing kinetics (13D). The error bars show standarddeviation and the black horizontal line marks the limit of detection.

FIG. 14. Is a graph showing the sensitivity of different bacterialspecies to P307 and P307SQ-8C. The error bars show standard deviationand the black horizontal line marks the limit of detection.

FIG. 15. FIGS. 15A and 15B are graphs that show the bactericidalactivities of P307 and P307SQ-8C against the log phase and stationaryphase of A. baumannii strain No. 1791 (15A) and the biofilm phase (15B).

FIG. 16. FIG. 16 shows the cytotoxic effects of P307 and P307SQ-8C asmeasured by B cell survival (16A) and hemolysis (16B).

FIG. 17. FIG. 17A shows the effect of DTT at 0, 0.1 and 1 mM on theactivity of P307 and P307SQ-8C. FIG. 17 B shows the effect ofsubstitution of the terminal cysteine residue of P307SQ-8C with alanine(P307SQ-8A).

FIG. 18. Is a DNA shift gel showing the shift for control peptide andP307.

FIG. 19. FIGS. 7 A-C show representative transmission electronmicroscopy images of A. baumanii strain no. 1791: untreated control(19A), treated with 300 μg/mL P307SQ-8C for 5 minutes (19B) and for 2hours (19C). Magnification, ×2600 (left, scale bar=2 μm) and ×5000(right top and bottom, scale bar=0.5 μm). FIG. 7D shows the bactericidalactivity of P307SQ-8C on gram negative bacteria K. pneumoniae and E.coli at pH 7.5 and 8.8.

FIG. 20. Shows the membrane permeability of A. baumannii strains #1791and S5 treated with P307 and P307SQ-8C.

FIG. 21. Shows the inhibition of bactericidal activity of P307 orP307SQ-8C by hydroxyl radical scavenger, thiourea and anaerobiccondition.

FIG. 22. Shows the effect of treatment of a skin infection withpolymyxin B and P307SQ-8C.

DETAILED DESCRIPTION

The present invention provides polypeptides having antibacterialactivity and for methods for using the disclosed polypeptides. As usedherein, the singular forms “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

Terms such as “comprises”, “comprised”, “comprising”, “contains”,“containing” and the like have the meaning attributed in United Statespatent law; they are inclusive or open-ended and do not excludeadditional, un-recited elements or method steps. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed in United States patent law; they allow for theinclusion of additional ingredients or steps that do not materiallyaffect the basic and novel characteristics of the claimed invention. Theterms “consists of” and “consisting of” have the meaning ascribed tothem in United States patent law; namely that these terms are closeended

In a first aspect, the invention provides polypeptides that comprise anamino acid sequence that has at least 90%, or at least 91%, or at least92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%,or at least 97%, or at least 98%, or at least 99%, or 100%, identity toSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21,or a fragment of the polypeptide, wherein the polypeptide or fragmenthas antibacterial activity.

In another embodiment of the first aspect, the polypeptides comprise anamino acid sequence that has at least 95%, or at least 96%, or at least97%, or at least 98%, or at least 99%, or 100%, identity to SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or afragment of the polypeptide, wherein the polypeptide or fragment hasantibacterial activity.

In yet another embodiment of the first aspect, the polypeptides comprisean amino acid sequence that has 100%, identity to SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or a fragment of thepolypeptide, wherein the polypeptide or fragment has antibacterialactivity.

In a second aspect, the invention provides polypeptides that consist ofan amino acid sequence that has at least 90%, or at least 91%, or atleast 92%, or at least 93%, or at least 94%, or at least 95%, or atleast 96%, or at least 97%, or at least 98%, or at least 99%, or 100%,identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, orSEQ ID NO:21, or a fragment of the polypeptide, wherein the polypeptideor fragment has antibacterial activity.

In another embodiment of the second aspect, the polypeptides consist ofan amino acid sequence that has at least 95%, or at least 96%, or atleast 97%, or at least 98%, or at least 99%, or 100%, identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or afragment of the polypeptide, wherein the polypeptide or fragment hasantibacterial activity.

In yet another embodiment of the second aspect, the polypeptidesconsists of an amino acid sequence that has 100%, identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21, or afragment of the polypeptide, wherein the polypeptide or fragment hasantibacterial activity.

In a third aspect, the invention provides polypeptides that comprise anamino acid sequence that has at least at least 80%, or at least 85%, orat least 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99%, or 100%, to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21, or a fragment of the polypeptide,wherein the polypeptide or fragment is conjugated to an antimicrobialpeptide to yield a conjugated polypeptide and the conjugated polypeptidehas antibacterial activity.

In one embodiment of the third aspect the polypeptide comprises an aminoacid sequence that has at least 90%, or at least 92%, or at least 94%,or at least 95%, or at least 96%, or at least 97%, or at least 98%, orat least 99%, or 100%, identity to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21, or a fragment of the polypeptide,wherein the polypeptide or fragment is conjugated to an antimicrobialpeptide to yield a conjugated polypeptide and the conjugated polypeptidehas antibacterial activity.

In a fourth aspect, the invention provides polypeptides that consists ofan amino acid sequence that has at least at least 80%, or at least 85%,or at least 90%, or at least 91%, or at least 92%, or at least 93%, orat least 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99%, or 100%, to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21, or a fragment of the polypeptide,wherein the polypeptide or fragment is conjugated to an antimicrobialpeptide to yield a conjugated polypeptide and the conjugated polypeptidehas antibacterial activity.

In one embodiment of the fourth aspect the polypeptide consists of anamino acid sequence that has at least 90%, or at least 92%, or at least94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%,or at least 99%, or 100%, identity to SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, or SEQ ID NO:21, or a fragment of the polypeptide,wherein the polypeptide or fragment is conjugated to an antimicrobialpeptide to yield a conjugated polypeptide and the conjugated polypeptidehas antibacterial activity.

In some embodiments of the third or fourth aspect, the antimicrobialpeptide comprises the amino acid sequence SQSRESQC (SEQ ID NO:44)wherein at least one amino is cysteine and 0, 1, 2, 3, 4, 5, 6, or 7amino acids of the antimicrobial peptide are conservatively substituted.0, 1, 2, 3, 4, 5, 6, or 7 amino acids of the antimicrobial peptide areconservatively substituted. In other embodiments of the third or fourthaspects, the antimicrobial peptide comprises the amino acid sequenceSQSRESQC (SEQ ID NO:44). In still other embodiments of the third orfourth aspect, the antimicrobial peptide comprises the amino acidsequence SQSRESQC (SEQ ID NO:44) wherein 0, 1, 2, 3, 4, 5, 6, or 7 aminoacids of the antimicrobial peptide are conservatively substituted andthe antimicrobial peptide consists of 8 amino acids. In yet otherembodiments of the third or fourth aspect, the antimicrobial peptideconsists of the amino acid sequence SQSRESQC (SEQ ID NO:44).

In some embodiments of the third or fourth aspect, the antimicrobialpeptide comprises the amino acid sequence CSQRQSES (SEQ ID NO:50)wherein at least one amino is cysteine and 0, 1, 2, 3, 4, 5, 6, or 7amino acids of the antimicrobial peptide are conservatively substituted.In other embodiments of the third or fourth aspects, the antimicrobialpeptide comprises the amino acid sequence CSQRQSES (SEQ ID NO:50). Instill other embodiments of the third or fourth aspect, the antimicrobialpeptide comprises the amino acid sequence CSQRQSES (SEQ ID NO:50)wherein 0, 1, 2, 3, 4, 5, 6, or 7, amino acids of the antimicrobialpeptide are conservatively substituted and the antimicrobial peptideconsists of 8 amino acids. In yet other embodiments of the third orfourth aspect, the antimicrobial peptide consists of the amino acidsequence CSQRQSES (SEQ ID NO:50).

In some embodiments of the third or fourth aspect, the C-terminus of thepolypeptide or the fragment is conjugated to the antimicrobial peptide.In other embodiments of the third or fourth aspect, the C-terminus ofthe polypeptide or the fragment is conjugated to the N-terminus of theantimicrobial peptide. In still other embodiments of the third or fourthaspect, the N-terminus of the polypeptide or fragment is conjugated tothe antimicrobial peptide. In yet other embodiments of the third orfourth aspect, the N-terminus of the polypeptide or fragment isconjugated to the C-terminus of the antimicrobial peptide. For any ofthe embodiments of the third or fourth aspect the antimicrobial peptidecan be conjugated to the polypeptide or fragment via a peptide bond.

Another embodiment of the peptides of the present disclosure is apeptide having the amino acid sequenceNAKDYKGAAAEFPKWNKAGGRVLAGLVKRRKSQSRESQA (SEQ ID NO: 53). Anotherembodiment is a peptide having the amino acid sequenceNAKDYKGAAAEFPKWNKAGGRVLAGLVKRRKCSQRQSES (SEQ ID NO:51).

In some embodiments the polypeptides, polypeptide fragments orconjugated polypeptides have antibacterial activity against agram-negative bacterium. In some embodiments, the gram-negativebacterium is of the genus Acinetobacter.

In some embodiments the polypeptides, polypeptide fragments orconjugated polypeptides have antibacterial activity against E. coli, P.aeruginosa or A. baumannii.

In some embodiments the polypeptides, polypeptide fragments orconjugated polypeptides have antibacterial activity against agram-positive bacterium. In some embodiments, the gram-positivebacterium is S. aureus or B. anthracis.

In some embodiments, the polypeptide is lyophilized.

Specific embodiments of the polypeptides of the invention are providedin Table 1.

TABLE 1 SEQ ID NO: 1 F307 SEQ ID NO: 2 F376 SEQ ID NO: 3 F351 SEQ ID NO:4 F347 SEQ ID NO: 5 F344 SEQ ID NO: 6 F340 SEQ ID NO: 7 F338 SEQ ID NO:8 F336 SEQ ID NO: 9 F334 SEQ ID NO: 10 F332 SEQ ID NO: 11 F330 SEQ IDNO: 12 F328 SEQ ID NO: 13 F324 SEQ ID NO: 14 F321 SEQ ID NO: 15 F320 SEQID NO: 16 F315 SEQ ID NO: 17 F306 SEQ ID NO: 18 F303 SEQ ID NO: 19 F301SEQ ID NO: 20 F309 SEQ ID NO: 21 F311 SEQ ID NO: 43 P307 SEQ ID NO: 44SQSRESQC SEQ ID NO: 45 P307SQ-8C (P307Ex) SEQ ID NO: 48 AEMLFLK SEQ IDNO: 49 P307AE-8 SEQ ID NO: 50 CSQRQSES SEQ ID NO: 51 P307CS-8 SEQ ID NO:52 SQSRESQA SEQ ID NO: 53 P307SQ-8A

P307SQ-8C and P307Ex are used interchangeably herein.

The invention also provides for pharmaceutical compositions comprisingthe polypeptides, polypeptide fragments or conjugated polypeptides ofthe invention. In some embodiments, the compositions are pharmaceuticalcompositions, which comprise a pharmaceutically acceptable carrier,buffering agent, or preservative.

In some embodiments, the pharmaceutical composition is formulated fortopical administration. In other embodiments, the pharmaceuticalcomposition is formulated for subcutaneous delivery. In still otherembodiments, the pharmaceutical composition is formulated forintravenous delivery. In yet other embodiments, the pharmaceuticalcomposition is formulated for oral delivery.

In some embodiments, the composition further comprises an antibiotic.Examples of suitable antibiotics include, but are not limited to,amoxicillin, augmentin, amoxicillin, ampicillin, azlocillin,flucloxacillin, mezlocillin, methicillin, penicillin G, penicillin V,cephalexin, cefazedone, cefuroxime, loracarbef, cemetazole, cefotetan,cefoxitin, ciprofloxacin, levaquin, and floxacin, tetracycline,doxycycline, or minocycline, gentamycin, amikacin, and tobramycin,clarithromycin, azithromycin, erythromycin, daptomycin, neomycin,kanamycin, or streptomycin.

In some embodiments, the pharmaceutical composition further comprises aclotting agent.

In some embodiments, the pharmaceutical composition is lyophilized.

The present invention also provides for methods for treating a subjectin need thereof, comprising administering to the subject apharmaceutical composition comprising a polypeptide, polypeptidefragment or conjugated polypeptide of the invention, and apharmaceutically acceptable carrier, buffering agent, or preservative.

In one embodiment the method is a method for treating a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising a polypeptide of the invention, and apharmaceutically acceptable carrier, buffering agent, or preservative.

In another embodiment the method is a method for treating a subject inneed thereof, comprising administering to the subject a pharmaceuticalcomposition comprising a polypeptide fragment of the invention, and apharmaceutically acceptable carrier, buffering agent, or preservative.

In one embodiment the method is a method for treating a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising a conjugated polypeptide of the invention, and apharmaceutically acceptable carrier, buffering agent, or preservative.

In one embodiment the method is a method for treating having a bacterialinfection and the treatment is therapeutic treatment comprisingadministering to the subject a pharmaceutical composition comprising aconjugated polypeptide of the invention, and a pharmaceuticallyacceptable carrier, buffering agent, or preservative. In someembodiments, the subject has a bacterial infection that isnon-responsive to other treatment modalities. For example, the bacterialinfection may be resistant to one or more antibiotic. In one embodiment,the bacterial infection is a wound infection.

In one embodiment the method is a method for prophylactically treating asubject in need thereof comprising administering to the subject apharmaceutical composition comprising a conjugated polypeptide of theinvention, and a pharmaceutically acceptable carrier, buffering agent,or preservative. In some embodiments the subject has undergone, or isundergoing surgery and the surgical wound is contacted with apharmaceutical composition of the invention. In certain embodiments, thesurgical wound is irrigated with the pharmaceutical composition prior toclosure of the wound. In other embodiments the pharmaceuticalcomposition is applied to the wound after closure, for example thepharmaceutical composition is applied to the sutured or stapled area ofthe wound.

In some embodiments, the method comprises administering a pharmaceuticalcomposition of the invention is administered in combination with anantibiotic. In some embodiments, the method comprises topicallyadministering a pharmaceutical composition of the invention. In otherembodiments, the method comprises administering a pharmaceuticalcomposition of the invention subcutaneously. In still other embodiments,the method comprises administering a pharmaceutical composition of theinvention by intravenous injection. In yet other embodiments, the methodcomprises administering a pharmaceutical composition of the inventionorally.

In some embodiments, the pharmaceutical composition is in a unit dosageform. In other embodiments, the pharmaceutical composition is in theform of a cream, ointment, salve, gel, lozenge, spray, or aerosol.

Also provided, are methods for treating a bacterial infection comprisinginhibiting the formation of or disrupting a bacterial biofilm comprisingadministering to a subject in need thereof, a composition comprising apolypeptide, polypeptide fragment or conjugated polypeptide of theinvention in an amount effective to kill bacteria in the biofilm.

Additionally provided, are methods of disinfecting an article comprisingcontacting the article with a composition comprising a polypeptide,polypeptide fragment or conjugated polypeptide of the invention to thearticle for a time sufficient to disinfect the article. In someembodiments, the article is a hard surface. In some embodiments, thearticle is a countertop, keyboard, surgical instrument, or medicaldevice.

Additionally provided, are methods for inhibiting the formation of ordisrupting a bacterial biofilm on an article comprising contacting thearticle with a polypeptide, polypeptide fragment or conjugatedpolypeptide of the invention, in an amount effective to kill bacteria inthe biofilm.

Also provided, are articles of manufacture that contain a compositioncomprising a polypeptide, polypeptide fragment or conjugated polypeptideof the invention. In some embodiments, the article of manufacture is aspray bottle that contains a polypeptide, polypeptide fragment orconjugated polypeptide of the invention.

In some embodiments, the article of manufacture contains apharmaceutical composition comprising a polypeptide, polypeptidefragment or conjugated polypeptide of the invention and a carrier,buffering agent or preservative. In some embodiments, the article ofmanufacture is a vial. In some embodiments, the article of manufactureis a delivery device. In some embodiments, the composition contained bythe article of manufacture is lyophilized.

Modifications and changes can be made in the structure of thepolypeptides of the disclosure and still obtain a molecule havingsimilar characteristics as the polypeptide (e.g., a conservative aminoacid substitution). For example, certain amino acids can be substitutedfor other amino acids in a sequence without appreciable loss ofactivity. Because it is the interactive capacity and nature of apolypeptide that defines that polypeptide's biological functionalactivity, certain amino acid sequence substitutions can be made in apolypeptide sequence and nevertheless obtain a polypeptide with likeproperties.

Such amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include(original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),(Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly:Ala), (His: Asn, Gln), (Be: Leu, Val), (Leu: Be, Val), (Lys: Arg), (Met:Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and(Val: Ile, Leu). Embodiments of this disclosure thus contemplatefunctional or biological equivalents of a polypeptide as set forthabove. In particular, embodiments of the polypeptides can includevariants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identityto the polypeptide of interest.

“Identity” as known in the art, is a relationship between two or morepolypeptide sequences, as determined by comparing the sequences.“Identity” can be readily calculated by known algorithms well known inthe art. Preferred methods to determine identity are designed to givethe largest match between the sequences tested. Methods to determineidentity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined using analysissoftware (i.e., Sequence Analysis Software Package of the GeneticsComputer Group, Madison Wis.) that incorporates the Needelman andWunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, andXBLAST).

Identity can be measured as “local identity” or “global identity”. Localidentity refers the degree of sequence relatedness between polypeptidesas determined by the match between strings of such sequences. Globalidentity refers to the degree of sequence relatedness of a polypeptidecompared to the full-length of a reference polypeptide. Unless specifiedotherwise, as used herein identity means global identity. Thepercentages for global identity herein are calculated using the ClustalWalgorithm used through the software MacVector, using the defaultsettings; both for local and global identity.

Production of Polypeptides

Polypeptides of the present invention can be produced by any knownmethod. For example, polypeptides can be produced in bacteria including,without limitation, E. coli, or in other existing system for polypeptide(e.g., Bacillus subtilis, baculovirus expression systems usingDrosophila Sf9 cells, yeast or filamentous fungal expression systems,mammalian cell expression systems), or they can be chemicallysynthesized.

If the a polypeptide is to be produced in bacteria, e.g., E. coli, thenucleic acid molecule encoding the peptide may also encode a leadersequence that permits the secretion of the mature peptide from the cell.Thus, the sequence encoding the peptide can include the pre sequence andthe pro sequence of, for example, a naturally occurring bacterial STpeptide. The secreted, mature peptide can be purified from the culturemedium.

The sequence encoding a peptide described herein is can be inserted intoa vector capable of delivering and maintaining the nucleic acid moleculein a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector may be abacterial or bacteriophage DNA vector such as bacteriophage lambda orM13 and derivatives thereof. Construction of a vector containing anucleic acid described herein can be followed by transformation of ahost cell such as a bacterium. Suitable bacterial hosts include but arenot limited to, E. coli, B subtilis, Pseudomonas, Salmonella. Thegenetic construct also includes, in addition to the encoding nucleicacid molecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art.The expression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or aninternal ribosome entry site). The vector can be capable of autonomousreplication or it can integrate into host DNA to ensure stability duringpeptide production.

One embodiment of a nucleic acid according to the present invention is anucleic acid that encodes a polypeptide comprising an amino acidsequence that has at least 90% sequence identity to the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, or SEQ ID NO:45, or a fragment of the polypeptide, whereinthe polypeptide or fragment has antibacterial activity.

In another embodiment, the nucleic acid encodes a polypeptide comprisingan amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:45, or a fragment of thepolypeptide, wherein the polypeptide or fragment has antibacterialactivity.

In yet another embodiment, the nucleic acid encodes a polypeptideconsisting of an amino acid sequence nucleic acid of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:45, or afragment of the polypeptide, wherein the polypeptide or fragment hasantibacterial activity.

In still another embodiment, the nucleic acid comprises the nucleotidesequence of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, or SEQ ID NO:42.

In still another embodiment, the nucleic acid consists of the nucleotidesequence of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ IDNO:41, or SEQ ID NO:42.

Another embodiment is an expression vector that comprises a nucleic acidthat encodes a polypeptide comprising an amino acid sequence that has atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ IDNO:45, or a fragment of the polypeptide, wherein the polypeptide orfragment has antibacterial activity.

In another embodiment, the expression vector comprises a nucleic acidthat encodes a polypeptide comprising an amino acid sequence of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ IDNO:45, or a fragment of the polypeptide, wherein the polypeptide orfragment has antibacterial activity.

In yet another embodiment, the expression vector comprises a nucleicacid that encodes a polypeptide consisting of an amino acid sequencenucleic acid of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, or SEQ ID NO:45, or a fragment of the polypeptide,wherein the polypeptide or fragment has antibacterial activity.

In still another embodiment, the expression vector comprises a nucleicacid that comprises the nucleotide sequence of SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.

In still another embodiment, the expression vector comprises a nucleicacid that consists of the nucleotide sequence of SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42.

Table 2 provides specific embodiments of the nucleic acids of theinvention showing the nucleotide SEQ ID NO: that corresponds to thepolypeptides they encode.

TABLE 2 Corresponding Nucleotide Amino Acid SEQ ID NO: SEQ ID NO: 22 123 2 24 3 25 4 26 5 27 6 28 7 29 8 30 9 31 10 32 11 33 12 34 13 35 14 3615 37 16 38 17 39 18 40 19 41 20 42 21

The nucleic acid that encodes a polypeptide described herein can also befused to a nucleic acid encoding a peptide affinity tag, e.g.,glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, myc tag or the influenza HA tag, in order tofacilitate purification. The affinity tag or reporter fusion joins thereading frame of the peptide of interest to the reading frame of thegene encoding the affinity tag such that a translational fusion isgenerated. Expression of the fusion gene results in translation of asingle peptide that includes both the peptide of interest and theaffinity tag. In some instances where affinity tags are utilized, DNAsequence encoding a protease recognition site will be fused between thereading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the polypeptides and variants described herein inprotein expression systems other than bacteria, and well known to thoseskilled in the art, can also be used to produce polypeptides in abiological system.

Polypeptides and variants thereof can be synthesized by the solid-phasemethod using an automated peptide synthesizer. For example, the peptidecan be synthesized on Cyc(4-CH₂Bxl)-OCH₂-4-(oxymethyl)-phenylacetamidomethyl resin using a doublecoupling program. Peptides can also be synthesized by many other methodsincluding solid phase synthesis using traditional FMOC protection (i.e.,coupling with DCC-HOBt and deprotection with piperdine in DMF).

Therapeutic and Prophylactic Compositions and their Use

This invention provides methods of treatment comprising administering toa subject in need thereof an effective amount of a polypeptide of theinvention. The subject is human or another animal, including but notlimited to primates such as monkeys and chimpanzees; livestock animalssuch as cows, pigs, horse or chickens; and companion animals such asdogs cats, and rodents. In a specific embodiment the subject is a human.In another specific embodiment the subject is a non-human mammal. In oneembodiment the polypeptides are administered as the sole antibacterialagent. In another embodiment the polypeptides are administered incombination with one or more other antibacterial agents.

Methods of administration of the disclosed pharmaceutical compositionscan be oral or parenteral and include but are not limited tointradermal, intramuscular, intraperitoneal, intravenous,intra-articular, intra-synovial, subcutaneous, intranasal, epidural,topical and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compositions of the invention into the central nervoussystem by any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent. In a specific embodiment, it may be desirable toadminister the pharmaceutical compositions of the invention locally tothe area in need of treatment, such as topical use on the skin; anysuitable method known to the art may be used.

In one aspect of the invention provides for pharmaceutical compositionscomprising the polypeptides of the present disclosure for therapeutic orprophylactic treatment of bacterial infections. An embodiment of theinvention is a pharmaceutical composition formulated for topicaltreatment. Another embodiment of the invention is a pharmaceuticalcomposition formulated for systemic infections.

Such compositions comprise a therapeutically effective amount of apolypeptide of the invention and a pharmaceutically acceptable carrier,buffering agent, or preservative. The term “pharmaceutically acceptablecarrier” as used herein, includes, but is not limited to, solvents,diluents, or other liquid vehicle, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, solid binders, lubricants and the like, as suited to theparticular dosage form desired. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition can also contain of wettingor emulsifying agents, preservatives, or pH buffering agents. Thesecompositions can take the form of a solution, suspension, emulsion,tablet, pill, lozenge, capsule, powder, patches for topicaladministration and the like. For topical applications, thepharmaceutically acceptable compositions may be formulated in a suitableointment, lotion or cream containing the active component suspended ordissolved in one or more carriers. Carriers for topical administrationinclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene-polyoxypropylenecompounds, emulsifying wax, polysorbate 60, cetyl esters wax, cetearyalcohol, 2-octyldodecanol, benzyl alcohol and water. The composition canbe formulated as a suppository with traditional binders and carrierssuch as triglycerides. Oral formulation can include standard carrierssuch as pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc. One ofskill in the art is well versed in formulation of therapeutic agents.See e.g. Remington The Science and Practice of Pharmacy, 20th Edition,Lippincott Williams & White, Baltimore, Md. (2000); Remington'sPharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).

The invention also provide a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) is a notice in the form prescribed by a governmentalagency regulating the manufacture, use, or sale of pharmaceuticals orbiologic products, which notice reflects (a) approval by the agency ofmanufacture, use or sale for human administration, (b) directions foruse, or both.

EXAMPLES

The following examples are put forth so as to provide additionalinformation to one of skill in the art of how to make and use thepolypeptides described herein, and are not intended to limit the scopeof what the inventors regard as their invention. Efforts have been madeto ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.) however, some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, molecular weight isaverage molecular weight, and the temperature is in degrees Centigrade.

Example 1

Identification of Polypeptides Having Antibacterial Activity.

Fifteen clinical isolates of A. baumannii were obtained from a New Yorkhospital. Strains of A. baumannii were isolated and treated withmitomycin C to induce prophage induction. The supernatants werecollected and phage were precipitated with polyethylene glycol (PEG).Supernatants from three of the A. baumannii isolates were examined bynegative staining EM and images taken of the phage (FIGS. 1A, 1B, and1C).

Phage DNA was separated from co-precipitated compounds by agarose gelelectrophoresis and extraction of high-molecular-weight DNA. From thisDNA, an expressible linker shotgun library (E-LASL) was constructed aspreviously described. (Schmitz J E. et al., 2008, Appl. Environ.Microbiol. 74:1649-1652.) Briefly, for all samples, 100 ng of DNA wasfragmented with the restriction enzyme TSP509I (consensus sequence AATT)Following phenol-chloroform extraction and ethanol precipitation, theDNA was ligated to 40 ng of linker sequence, with a complementary 5′overhang (AATTCGGCTCGAG, where the overhang is underlined (SEQ IDNO:46). The ligation mixture was used as the template for Taq-based PCRusing the linker-targeted primer CCATGACTCGAGCCGAATT (SEQ ID NO:47).

The amplified inserts were ligated into the arabinose-inducible pBADplasmid using the pBAD TOPO® TA expression kit; Invitrogen, per themanufacturer's directions. The recombinant vectors were transformed intocompetent E. Coli TOP10 (Invitrogen). To determine which clones hadlytic activity, the E. coli were plated on LB agar supplemented with 100μg/ml ampicillin and 5% defibrinated sheep's blood. Following overnightgrowth at 37° C., the plates were placed in a sealed container that wasattached to the outlet of a commercial nebulizer. Nebulized arabinosewas continuously pumped into the container for 1 hour. The plates werereturned to 37° C. and colonies were identified that developed a zone ofhemolysis in the surrounding blood agar. Chosen clones were streakedonto separate LB-ampicillin plates (lacking arabinose) and allowed topropagate without induced expression. (Schmitz J E, et al., 2010 Appl.Environ. Microbiol. 76:7181-7187).

To determine killing activity for A. baumannii, a secondary screen wasdone essentially as described in Schmitz J E, et al., 2010 Appl.Environ. Microbiol. 76:7181-7187. Hits were streaked as approximately1-cm by 2-cm patches onto LB-ampicillin plates supplemented with 0.2%arabinose. Following overnight incubation at 37° C., the plates wereexposed to chloroform vapor to kill and permeabilize any still-viable E.coli. The patches were then overlaid with molten soft agar containing A.baumannii and observed for clearing zones. Twenty-one positive cloneswere identified that exhibited a clear zone around the clone. FIG. 2shows a representative screen of lysin clone activity in clearing liveA. baumannii imbedded in the agar.

The inserts of the positive closes were sequenced and compared to thesequences in the NCBI protein database. The alignments showed that amongthe 21 clones there are four classes of lytic activity: i) nine were inthe glycosyl hydrolase family, ii) seven were phage baseplate lysozymes,iii) two were lysozyme autolysins, and iv) three were lysins. (FIG. 3).For ease of reference here in, regardless of class, the polypeptidesencoded by these sequences are referred to as “lysins”. FIG. 4 shows asequence alignment based on similarity of the nucleotide sequencesencoding the 21 clones. FIG. 5 shows a sequence alignment based onsimilarity of the polypeptide sequences of the 21 clones.

Example 2

Activity of Positive Clones.

Twenty-one different constructs were screened for activity againstthirteen different A. baumannii clinical isolates. The constructs wererecombinantly expressed in E. coli. Cells were grown at 30° C. 200 rpm,and when reaching mid-log phase they were induced by adding 0.2%arabinose. Induction continued overnight. In the morning, cells werespun down, washed 3× with 50 mM sodium phosphate buffer pH 7.0, beforebeing homogenized in an Emulsiflex homogenizer. Cell debris was removedby centrifugation (16000 g, 45 min) and the lysate passed through a 0.22um sterile filter to generate the crude lysate.

A. baumannii grown overnight in TSB, were mixed with 50° C. soft agarTSB and poured onto a TSB agar plate as a top agar layer. The plate wasallowed to solidify in room temperature. Crude lysates (10 ul) wereadded to a soft agar plate with A. baumannii, and incubated for 2 hoursat room temperature each day, while being kept at 4° C. for theremainder of the time. Plates were incubated until clearing zones werevisible (4-5 days). A clearing zone larger than the original spot of thecrude lysate was scored. The number above each lysin indicates how manystains for which that lysin was the most efficient.

Results are shown in FIG. 6. The lysin construct is shown on the x-axisand the percent of Acinetobacter strains lysed is shown on the y-axis.The numbers above each bar indicates the number of strains for whichthat lysin was the most efficient, no number indicates one strain. Ascan be seen, Lysin F307 lysed about 90% of the strains tested and wasthe most active against seven strains.

Example 3

Lysis of A. baumannii by F307.

FIG. 7 shows representative Transmission Electron Micrographs of A.baumannii strain 1791 cells after treatment with F307 polypeptide. Themicrographs show that F307 caused lysis through extrusion of thecytoplasmic membrane to the outside of the cell. (See FIG. 7, arrows).Two 100 mL culture of A. baumannii strain 1791 were started in BHI mediaand grown in a 500 mL flask for 1.5 h at 37° C., 200 rpm. The cells werethen centrifuged and washed one time with 1×PBS buffer. They were thenre-suspended in 1.2 mL of 1×PBS. EDTA at a final concentration of 250 μMwas added to each sample. To the experimental sample 300 μL of lysin(˜1.2 mg final concentration) and incubated the control (EDTA alone) andexperimental (EDTA+F307 lysin) at 25° C. Time points were taken at 0.5,1, 5, 10, 15, and 30 minutes. The reactions were quenched and cells werefixed using 2.5% Gluteraldhyde in CAC buffer (10 mm sodium cacodylate,0.1 m CaCl2, pH 6.5).

Example 4

Effect of F307 Polypeptide on A. baumannii Biofilms on Catheters InVitro and In Vivo.

In Vitro Treatment of Catheter Adherent A. baumannii 1791 with F307Lysin

Catheter tubing (CareFusion Ref#72023E) was cut using a sterile scalpelinto 3-inch long sections. An overnight culture of A. baumannii 1791 wasused to inoculate 1:1000 a 50 mL of TSB 0.2% Glucose (˜1×105 CFU/mL).Each 3-inch catheter tube was seeded with 300-350 of the 1:1000 dilutedculture. The catheters were then clamped and placed in plasticcontainers in a 37° C. incubator for 3 days to allow for biofilmformation to occur. After the 3 days catheters were washed twice witheither PBS or Sodium Phosphate buffer pH 7.5 and then had 300-350 μL ofF307 added to the tube (˜1 mg final concentration). The catheters werethen clamped. Catheters were taken at time points 0, 15 minutes, 30minutes, and 1 hour. The catheters were washed twice with 50 mM SodiumPhosphate pH 7.5 and were cut into small pieces. These were placed intoa 1.5 mL ependorff tube and 500 μL of 50 mM NaP buffer pH 7.5 was added.The tubes were sonicated for 20 minutes, and vortexed for 1 minute. Thesamples were then serial diluted and 20 μL was plated onto a quadrant ofa BHI agar plate and incubated at 37° C. overnight. CFU's werecalculated the following morning.

An approximate 4-log drop in the number of colony forming units (CFU'sof A. baumannii was observed after 30 minutes of treatment. Table 3shows CFU counts. FIG. 8 shows Scanning Electron micrographs of 3-daybiofilms of A. baumannii strain 1791 before and after treatment with 250μg F307 polypeptide.

TABLE 3 Treatment of A. baumannii biofilms on catheters. Sample CFU notreatment 1.4 × 10⁷ no treatment replicate 3.0 × 10⁶ 15 minutes F307treatment 9.0 × 10⁴ 30 minutes F307 treatment 6.0 × 10³

Example 5

Mouse Catheter Model: Several 3 inch section of catheter tubing wereseeded (1:1000) with A. baumannii strain 1791. A. baumannii biofilmswere formed as described above. The back of twenty BALB/C mice wereshaved, their backs were sterilized, and then an incision was made toplace a 1 inch section of the catheter with a biofilm already formedunder the dermis of the back. The incisions were then stapled shut.After 24 hours, 250 μl of F307 (1 mg) (n=10) or 250 μl control vehicle(n=10) was injected directly into the catheter that was under the dermisof the mouse. The treatment was repeated after 4 hours. After 3 hoursthe catheters were removed from the mice, and assayed as described inExperiment 4. FIG. 9 shows the reduction of bacterial counts byapproximately 2-logs in mice treatment with F307 polypeptide comparedwith control.

Example 6

F307 Polypeptide Rescues Mice from Death after a Lethal Injection of A.baumannii.

Twenty-two C57BL/6 mice were given 10⁸ CFU of A. baumannii strainintraperitoneally (IP). Two hours later, two mice were euthanized andorgans examine as described below, ten mice injected IP with 1 mg ofF307 and ten mice were injected IP with control vehicle. Treated animalsshowed 50% survival with this dose of lysin, whereas control mice showedonly 10% survival 14 days after infection (FIG. 10).

The organs from the two mice that were euthanized after infection wereexamined to confirm that the organs were infected with A. baumannii atthe time of treatment with F307 polypeptide. Liver, spleen, kidney, andheart were dissected from the mice. The organs were then homogenized in500 μl of 1×PBS. Dilutions were made and were plated onto Brain Heartinfusion (BHI) plates. The plates were incubated at 37° C. overnight.The number of colony forming units was counted. Control mice weresacrificed at the two hour time point and showed Acinetobacter in allorgans examined indicating that the organs were infected with A.baumannii at the time of treatment.

Example 7

P307 polypeptide (SEQ ID NO:43) was tested in duplicate against 18clinical isolates of A. baumannii strains. A. baumannii strains werecultured ON to reach stationary phase. Cells were washed 3× in 20 mMTris pH 7.5 and resuspended in the same buffer to an OD (595 nm) ofaround 0.7. To these cells, P307 (250 ug/ml) or a corresponding volumeof buffer, was added, and the mixture was allowed to incubate for 60minutes at room temperature. Dilutions of the mixtures were made andplated on TSB Agar plates for subsequent counting of colony formingunits.

P307 polypeptide treatment resulted in a 1 to 8-log drop in bacterialviability, versus control, after incubation for 60 minutes with 250 μgof P307. Results are show in Table 4. When P307 was compared with thefull length F307 polypeptide (SEQ ID NO:1) the P307 polypeptide hadhigher activity.

TABLE 4 P307 activity against 18 A. baumannii strains. Control P307 LogStrain Control 1 Control 2 Average P307 1 P307 2 Average Difference drop1775 1.00E+08  4.50E+08  2.75E+08  1.00E+07  1.50E+07  1.25E+07 2.20E+01  1.34 1776 5.50E+08  3.50E+08  4.50E+08  8.80E+05  7.50E+05 8.15E+05  5.52E+02  2.74 1777 7.00E+08  4.00E+08  5.50E+08  6.50E+06 9.00E+06  7.75E+06  7.10E+01  1.85 1788 2.00E+08  3.00E+08  2.50E+08 1.50E+07  1.20E+07  1.35E+07  1.85E+01  1.27 1789 4.50E+08  3.50E+08 4.00E+08  1.10E+07  1.30E+07  1.20E+07  3.33E+01  1.52 1790 1.50E+08 2.00E+08  1.75E+08  5.50E+05  1.80E+05  3.65E+05  4.79E+02  2.68 17919.0E+08 4.5E+08 6.8E+08 2.2E+05 2.2E+05 2.2E+05 3.1E+03 3.49 17921.2E+09 8.5E+08 1.0E+09 7.1E+05 7.5E+05 7.3E+05 1.4E+03 3.15 17933.5E+08 5.0E+08 4.3E+08 6.5E+05 5.6E+05 6.1E+05 7.0E+02 2.85 17947.5E+08 4.0E+08 5.8E+08 7.0E+05 6.0E+05 6.5E+05 8.8E+02 2.95 17959.5E+08 1.3E+09 1.1E+09 9.0E+06 2.5E+07 1.7E+07 6.6E+01 1.82 17961.0E+09 7.0E+08 8.5E+08 8.2E+05 8.2E+05 8.2E+05 1.0E+03 3.02 17971.2E+09 9.0E+08 1.1E+09 6.7E+05 6.5E+05 6.6E+05 1.6E+03 3.20 17984.0E+08 4.0E+08 4.0E+08 2.7E+05 6.5E+05 4.6E+05 8.7E+02 2.94 17995.5E+08 3.5E+08 4.5E+08 2.9E+07 7.0E+06 1.8E+07 2.5E+01 1.40 S1 1.4E+091.1E+09 1.3E+09 4.2E+07 3.0E+07 3.6E+07 3.5E+01 1.54 S3 2.5E+08 2.0E+082.3E+08 6.8E+05 6.5E+05 6.7E+05 3.4E+02 2.53 S5 1.1E+09 8.5E+08 9.8E+081.0E+00 1.0E+00 1.0E+00 9.8E+08 8.99

Example 8

Addition of a Short Extension Peptide Resulted in IncreasedAntibacterial Activity of P307.

The peptide SQSRESQC (SEQ ID NO:44) is derived from hepatitis C virusand has been shown to have antimicrobial activity against gram-positiveand gram-negative bacteria. We conjugated this sequence to P307 (P307Ex)to determine its effect on the activity. The sequence of F307, p307 andthe P307Ex (SEQ ID Nos: 1, 43 and 45 respectively) are provided in FIG.11 where a portion of the sequence of F307 is underlined to show thelocation of P307 and a portion of the sequence of P307 is doubleunderline to show the location of the antimicrobial sequence.

P307 and P307Ex were assayed in duplicate against six bacterial strains.Antibacterial acidity was measured as described in Example 5. Treatmentwith P307Ex resulted in a 3.2 log drop in A. baumannii 1791 whereastreatment with P307 resulted in a 2.9 log drop demonstrating that theaddition of the antimicrobial peptide increased the activity of P307.The results are shown in Table 5.

TABLE 5 Strain Control 1 P307 EX1 P307 EX 2 P307Ex Average DifferenceLog drop 1775 5.00E+08 1.60E+05 1.10E+05 1.35E+05 3.70E+03 3.5 17765.00E+08 5.50E+05 6.50E+05 6.00E+05 8.33E+02 2.9 1777 6.50E+08 6.50E+042.80E+05 1.73E+05 3.77E+03 3.5 1788 3.50E+08 8.80E+05 5.80E+05 7.30E+054.79E+02 2.6 1789 4.00E+08 1.10E+07 1.30E+07 1.20E+07 3.33E+01 1.5 17902.00E+08 1.50E+04 2.00E+04 1.75E+04 1.14E+04 4.0 1791 3.50E+08 4.00E+044.50E+04 4.25E+04 8.24E+03 3.9 1792 1.00E+08 4.00E+04 5.00E+03 2.25E+044.44E+03 3.6 1793 1.50E+08 3.50E+04 2.00E+04 2.75E+04 5.45E+03 3.7 17945.00E+07 1.40E+05 1.00E+05 1.20E+05 4.17E+02 2.6 1795 4.00E+08 5.50E+041.30E+05 9.25E+04 4.32E+03 3.6 1796 2.50E+08 3.80E+05 2.50E+05 3.15E+057.94E+02 2.8 1797 2.50E+08 5.50E+06 8.50E+06 7.00E+06 3.57E+01 1.5 17983.50E+08 3.40E+05 3.70E+05 3.55E+05 9.86E+02 3.0 1799 3.50E+08 5.00E+033.00E+04 1.75E+04 2.00E+04 4.3 S1 8.50E+08 5.90E+05 7.00E+05 6.45E+051.32E+03 3.1 S3 3.00E+08 1.60E+07 1.40E+07 1.50E+07 2.00E+01 1.3 S51.50E+09 5.00E+05 2.90E+05 3.95E+05 3.80E+03 3.57

P307 and P307Ex were tested for activity against A. baumannii strain1791, E. coli, P. aeruginosa strain PAO1, S. aureus strain RN4220, S.aureus strain 8325 and B. anthracis. As shown in Table 6, P307 and P307were most active against A. baumannii and B. anthracis.

TABLE 6 P307 and P307Ex against other bacterial species. Pseudomonas A.baumannii B. anthracis aeruginosa S. aureus S. aureus Sample 1791 E.coli Δsterne PAO1 RN4220 8325 control 1 5.50E+08 5.50E+08 2.40E+071.30E+09 1.50E+09 6.50E+08 control 2 2.60E+08 4.50E+08 2.80E+07 4.50E+087.50E+08 9.50E+08 P307EX 1 1.10E+05 3.50E+08 2.60E+03 4.70E+07 5.20E+073.20E+07 P307EX 2 3.90E+05 3.00E+08 2.90E+03 3.70E+07 5.80E+07 3.60E+07P307 1 5.80E+05 3.50E+08 3.10E+03 5.60E+07 8.00E+08 4.80E+07 P307 23.70E+05 3.50E+08 4.00E+03 4.00E+07 4.50E+08 4.40E+07 average control4.05E+08 5.00E+08 2.60E+07 8.75E+08 1.13E+09 8.00E+08 average P307EX2.50E+05 3.25E+08 2.75E+03 4.20E+07 5.50E+07 3.40E+07 average P3074.75E+05 3.50E+08 3.55E+03 4.80E+07 6.25E+08 4.60E+07 difference1.62E+03 1.54E+00 9.45E+03 2.08E+01 2.05E+01 2.35E+01 P307EX differenceP307 8.53E+02 1.43E+00 7.32E+03 1.82E+01 1.80E+00 1.74E+01 log drop 3.20.2 4.0 1.3 1.3 1.4 P307EX log drop P307 2.9 0.2 3.9 1.3 0.3 1.2

Example 9

P307 is not Toxic to B Cells or Red Blood Cells.

P307 was mixed with red blood cells to determine if it would causelysis. No lysis was observed at 200 μg of P307. When P307 was tested forlysis of a B cell line it was found to have only a slight effect on cellnumber after 24 hours. The results are shown in Table 7.

TABLE 7 (% viable) Sample 0 min 5 min 30 min 1 hour 2 hour 3 hour 24hours Initial cell only 97% 83.60%   85% 88.20% Tris-HCl pH = 6.8 97.20%89.70% 94.70% 81.50% 90.90% 89.90% 200 μg P307 94.60%  1100% 71.40%71.90% 58.60% 76.30%  20 μg P307 97.20% 88.60% 89.70% 90.30% 91.00%94.20%  2 μg P307 95.20% 76.90% 89.30% 92.50% 93.80% 96.30%

The peptides used in Examples 10-20 were chemically synthesized.

Peptides were created using a Protein Technologies Symphony™ peptidesynthesizer (PTI Tucson, Ariz., USA) on pre-coupled Wang(p-alkoxy-benzyl alcohol) resin (Bachem, Torrance, Calif., USA).Reaction vessels were loaded at 25 μM and peptides were elongated usingFmoc protected amino acids (Anaspec, San Jose, Calif., USA) (1997.Standard Fmoc protocols. 289:44-67). Deprotection of the amine wasaccomplished with 20% piperidine (Sigma-Aldrich) in NMP(N-methylpyrrolidinone). Repetitive coupling reactions were conductedusing 0.6 M HATU/Cl-HOBT (azabenzotriazol tetramethyluroniumhexafluorophosphate/6-chloro-1-hydroxybenzotriazole) (P3 Biosystems,Shelbyville, Ky., USA) and 0.4 M NMM (N-methylmorpholine) using NMP(EMD) as the primary solvent (1989. New Coupling Reagents in PeptideChemistry 30:1927-1930.). Resin cleavage and side-chain deprotectionwere achieved by transferring to a 100 ml round bottom flask and reactedwith 4.0 ml concentrated, sequencing grade, trifluoracetic acid (Fisher)with triisopropylsilane (Fluka), degassed water, and3,6-dioxa-1,8-octanedithiol (DODT, Sigma-Aldrich) in a ratio of 95:2:2:1over a 6 hour time frame. This was followed by column filtration to a 50ml round bottom flask and TFA volume reduced to 2 ml using a rotaryevaporator. A standard ether precipitation was performed on theindividual peptides by transferring to a 50 ml falcon tube containing 40ml cold tert-butyl methyl ether (TBME, Sigma-Aldrich). Samples wereplaced in an ice bath for 2 hours to aid precipitation followed bypellet formation using centrifugation (3300 rpm, 5 min). Excess etherwas removed by vacuum aspiration and the peptide pellets were allowed todry overnight in a fume hood. Dried peptide pellets were resolved in 20%acetonitrile and 10 ml HPLC grade water, subsampled for LC/MS andlyophilized. All crude products were subsequently analyzed byreverse-phase Aquity™ UPLC (Waters Chromatography, Milford, Mass., USA)using a Waters BEH C18 column. Individual peptide integrity was verifiedby tandem electrospray mass spectrometry using a ThermoFinnigan LTQ™(Thermo Fisher, Waltham, Mass., USA) spectrometer system. Preparativechromatography was accomplished on a Vydac C18 RP preparative column ona Waters 600 Prep HPLC. Individual fractions were collected in 30seconds intervals, characterized using LC/MS and fractions containingdesired product were lyophilized. These were stored at −20° C. untilbeing resuspended in autoclaved Milli-Q water for various assays. Thestock solutions were then stored at 4° C. The peptides are summarizedwith their amino acid sequences, isoelectric points (pI) and molecularweights (MW) in table 8.

TABLE 8 SEQ ID Names Amino acid sequences pI MW NO: F307 10.12  16 kDa 1P307 NAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK 10.71 3.4 kDa 43 P307AE-8NAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK AEMELFLK 10.21 4.4 kDa 49 P307SQ-8CNAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK SQSRESQC 10.38 4.3 kDa 45 P307C5-8NAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK CSQRQSES 10.38 4.3 kDa 51 P307SQ-8ANAKDYKGAAAEFPKWNKAGGRVLAGLVKRRK SQSRESQA 10.69 4.3 kDa 53

Example 10

Comparison of In Vitro Bactericidal Activities of Peptides of thePresent Disclosure.

To determine the in vitro bactericidal activities of the peptides, P307,P307AE-8, P307SQ-8C, and P307CS-8, bacteria were treated with thepeptides for 2 hours at room temperature. The survived cells wereserially diluted and plated on TSB agar plates to determine theactivity.

The bactericidal activities of 50 μg/mL the peptides, P307, P307AE-8 andP307SQ-8C were compared by treating A. baumannii strains #1791, S5 andATCC17978. P307SQ-8C was the most active, reducing about 10⁶ cfu/mL ofbacteria to below the limit of detection (<10 cfu/mL). P307 was slightlymore active than P307AE-8, but both peptides induced about a3.8-log-unit decrease in viable bacteria (FIG. 2A). To investigate howthe eight amino acids, SQSRESQC, contributed to the higher activity ofP307SQ-8C, the same molar concentration of peptide SQSRESQC as 50 μg/mLP307 was added by itself or in combination with P307 to A. baumanniistrains #1791 and S5. The activities were compared with 50 μg/mL of P307and P307SQ-8C. The combination was only as active as P307 while SQSRESQCpeptide alone has no activity (FIG. 2B). Hence the linkage is essentialfor the high bactericidal activity of P307SQ-8C. Next, we investigatedthe importance of sequence and composition. By scrambling the last eightamino acids in P307SQ-8C, we synthesized P307CS-8 with a C-terminaladdition of CSQRQSES to P307. The activities of P307SQ-8C and P307CS-8were comparable (FIG. 2C). The error bars show standard deviation andthe black horizontal line marks the limit of detection. Thus, weconcluded that the superior activity of P307SQ-8C derives from thecomposition of the last eight amino acids, regardless of the order ofthe last eight amino acids. For further investigation, we used P307SQ-8Cbecause it is the most active, and compared its activity with P307.

Example 11

Bactericidal Activities of P307 and P307SQ-8C

The effects of pH and NaCl on the in vitro activities of P307 andP307SQ-8C were investigated. A. baumannii strain #1791 were treated with50 μg/mL of peptides to test each condition. Two buffer systems (sodiumphosphate and Tris-HCl) were used to test pH 6.8, 7.5, 8.0 and 8.8. Thepeptides were more active in Tris-HCl and higher pH elicited betterkilling (FIG. 13A). Thus, we elected to continue our in vitroexperiments with 50 mM Tris-HCl, pH 7.5, which approximatesphysiological pH. The activities of both peptides were inverselyproportional to the concentration of NaCl (FIG. 13B). Next, titration ofP307 and killing kinetics of P307 and P307SQ-8C were investigated bytreating A. baumannii strain #1791. The activity of P307 wasconcentration-dependent, beginning from 4 μg/mL (FIG. 13C). P307SQ-8Cacted faster than P307, resulting in about 3.2-log-unit decrease alreadyat the 5 minute time point (FIG. 13D). There was no difference inactivities of either peptide at room temperature or 37° C. (data notshown). From these in vitro characterization experiments, we decided ouroptimal experimental conditions to be 50 mM Tris-HCl, pH 7.5, 50 μg/mLpeptides and 2 hours at room temperature (22-25° C.), unless otherwiseindicated.

Example 12

Next, we investigated the in vitro bactericidal spectra of P307 andP307SQ-8C against different bacterial species, A. baumannii (strain Nos.1775, 1776, 1777, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1796, 1797,1798, 1799, ATCC 17978 and S1, S3, D5), Bacillus anthracis (ΔSterne),Escherichia coli (DH5α), Pseudomonas aeruginosa (PA01), Staphylococcusaureus (RN4220) and two strains of Klebsiella pneumonia (ATCC 700603 andATCC10031). These bacterial species were treated with 50 μg/mL of P307or P307SQ-8C in 50 mM Tris-HCl, pH 7.5 for 2 hours at room temperatureto investigate the specificity of the peptides. Among the bacteriatested, A. baumannii strains were consistently most sensitive to thepeptides, showing an average of 2.7- and 6.2-log-unit decrease with P307and P307SQ-8C, respectively. Bacillus anthracis, Pseudomonas aeruginosaand Staphylococcus aureus are moderately sensitive. P307 and P307SQ-8Cproduced an average of about 1.3- and 2.9-log unit decrease,respectively, for these bacteria. However, Escherichia coli andKlebsiella pneumoniae are resistant to both peptides (FIG. 14).

Example 13

In addition, to investigate the activities of the peptides against A.baumannii at different growth phases, we compared the sensitivities ofstrain #1791 at log phase, stationary phase and biofilm state. Thebacteria in log phase (3 hours post inoculation of 1:100 overnightculture in fresh media) and stationary phase (overnight culture) weretreated with 50 μg/mL of P307 or P307SQ-8C for 2 hours at roomtemperature. The survived cells were serially diluted and plated on TSBagar plates to determine cfu/mL. (FIG. 15A). A. baumannii biofilms wereestablished by incubating about 10⁵ cfu/mL of strain #1791 in TSB with0.2% glucose inside about 2.5 cm long catheters for 72 hours at 37° C.The catheters were then washed to remove planktonic cells and treatedwith 250 μg/mL of P307 or P307SQ-8C. After 2 hours and 24 hours at roomtemperature, the biofilm was thoroughly disrupted and survived cellsre-suspended to be plated and counted to determine the killingefficiency of the peptides against in vitro biofilm (FIG. 15B) The logphase organisms were slightly more sensitive to P307 than stationaryphase (about 3.7—versus 2.4-log-unit decrease). There seems to be nosuch difference with P307SQ-8C (FIG. 15A). The biofilms were the mostresistant of all growth phases. Biofilms were treated with 250 μg/mLP307 or P307SQ-8C for 2 or 24 hr. After 2 hours, about 3- and 4-log-unitdecrease in cfu/mL was observed with P307 and P307SQ-8C, respectively.After 24 hours, P307 produced an additional about 1.3-log-unit decreasewhile P307SQ-8C did not (FIG. 15B).

Example 14

In order to compare the efficiency of the peptides P307 and P307SQ-8Cwith some clinically used antibiotics, we performed a minimal inhibitoryconcentration assay for two A. baumannii strains, #1791 and ATCC17978.Microtiter dilution method was used to determine the MICs oflevofloxacin, ceftazidime, polymyxin B, P307 and P307SQ-8C for A.baumannii strains #1791, #1798, S5 and ATCC17978. For the antibiotics,1.5-2 fold serial dilutions (three lower and three higher) of the MICsdetermined by Etest Lood R, et al., 2015 Antimicrob. Agents Chemother.59:1983-1991) were included. For the peptides, two-fold serial dilutions(500-31.25 μg/mL) were tested. The overnight cultures were re-suspendedto OD₆₀₀ of 0.001 (about 10⁵ cfu/mL) in Mueller-Hinton broth (pH 7.9).The antibiotics or peptides were added to final 100 μL for eachdilution. The bacteria were allowed to grow at 37° C. for 24 hour at 220rpm. The absorbance at 595 nm was then read in a SpectraMax Plus Reader(Molecular Devices). The MICs were determined as the lowestconcentrations of antimicrobial agents that completely inhibit bacterialgrowth. Alamar®Blue was used to confirm the data obtained from OD₅₉₅.The experiments were conducted at least twice in duplicate.

The strains displayed varying degree of sensitivity to all antimicrobialagents (Table 9).

TABLE 9 A. baumannii Levofloxacin Ceftazidime Polymyxin B P307 P307SQ-8Cstrains μg/mL μM μg/mL μM μg/mL μM μg/mL μM μg/mL μM #1791 6 16.6 250457 0.25 0.19 375 110 125 29 ATCC17978 ≤0.1 0.3 12 21.9 0.25 0.19 750220 ≤500 ≤116

P307SQ-8C has a lower MIC than P307, which is in accordance with the invitro killing activity (FIGS. 2 and 3).

Example 15

Cytotoxic Effects of P307 and P307SQ-8C as Measured by B Cell Survivaland Hemolysis.

Human B-cells obtained from a rheumatic fever patient at The RockefellerUniversity Hospital were grown in RPMI media supplemented with 10%bovine serum, penicillin and streptomycin. Cells were harvested by lowspeed centrifugation, washed once in media, and resuspended inpre-warmed media to a concentration of 10⁷ cells/ml, as determined bytrypan blue exclusion tests. The peptides (P307, P307SQ-8C and melittin)were serially diluted (80-0.3125 μM) in culture media, and added to5×10⁴ live cells. Cells were incubated for 1 hour at 37° C. in ahumidified 5% CO₂ atmosphere, after which they were stained (CellTiter96 Non-radioactive cell proliferation assay; Promega) according tomanufacturer's instructions. The samples were incubated for additionally4 hours, before a Solubilization/Stop solution was added, and incubatedovernight. The absorbance at 570 nm was measured in SpectraMax PlusReader (Molecular Devices). The reactions were carried out twice intriplicate and representative data are shown as mean±standard deviation.

Human blood from a healthy individual was gathered in an EDTA-tube, andred blood cells (RBC) collected through low speed centrifugation. Cellswere washed in PBS, and resuspended to a 10% RBC solution. P307 andP307SQ-8C were serially diluted in PBS (80-0.3125 μM). PBS and 1% TritonX-100 were used as negative and positive controls, respectively. Sampleswere mixed, and incubated for 1 hour at 37° C. The supernatant wascollected, and absorbance at 405 nm recorded through SpectraMax PlusReader (Molecular Devices). The reactions were carried out twice intriplicate and representative data are shown as mean±standard deviation.

Serial dilutions of peptides were incubated with about 5×10⁴ live Bcells for 1 hr at 37° C. in a humidified 5% CO₂ atmosphere, and melittinwas used as a positive control. CellTiter 96® Non-Radioactive CellProliferation Assay (Promega) was conducted according to manufacturer'sprotocol to quantify the survival of B cells. Red blood cells (RBCs)were incubated with serial dilutions of the peptides and the release ofhemoglobin into the supernatant was measured by OD₄₀₅ to determinehemolysis. Triton X-100 was used as a positive control. The error barsshow standard deviation.

The peptides were tested for their cytotoxicity using human B cells andred blood cells (RBCs). In contrast to the melittin positive control,the membranes of B cells are not affected by either P307 or P307SQ-8C.Even at the highest concentration tested (80 μM), the viability of thecells remains the same as the buffer control (FIG. 16A). Similarly, theintegrity of RBCs are also not affected by either peptide in comparisonto the Triton X-100 positive control (FIG. 16B).

Example 16

A portion of P307SQ-8C (about 25%) runs at twice the theoreticalmolecular weight in comparison to P307SQ-8A, which runs at 4.3 kD (datanot shown). To determine the importance of disulfide bond formation forthe high activity of P307SQ-8C the bactericidal activities of P307 andP307SQ-8C were compared in the presence of 0, 0.1 and 1 mMdithiothreitol (DTT). A. baumannii strain #1791 was treated with 50μg/mL P307 or 10 μg/mL P307SQ-8C in 50 mM Tris-HCl, pH 7.5 for 2 hoursat room temperature. The survived cells were serially diluted and platedon TSB agar. P307SQ-8C becomes less active with higher DTT concentrationwhereas the activity of P307 slightly increases (FIG. 17A). To furtherconfirm the importance of disulfide formation for P307SQ-8C activity, wesynthesized P307SQ-8A with the last cysteine changed to alanine. A.baumannii strains no. 1791 and ATCC17978 were treated with 10 μg/mL ofeach peptide. The bactericidal assays of P307SQ-8C and P307SQ-8A showedthat the former is slightly more active than the latter (FIG. 17B).These results altogether pointed out that part of the superior activityof P307SQ-8C derives from disulfide bond formation between twomolecules.

Example 17

Next, we investigated whether P307 binds to DNA, given the positivecharges on the peptides (net charge of +7). The peptide P307 was mixedwith DNA at different peptide:DNA ratios (0:1-15:1) and incubated for 1hour before being analyzed on an agarose gel. In comparison to positivecontrol peptide, no shift in molecular weight was observed for P307 atany of the ratios of peptide to DNA tested (FIG. 18).

Example 18

Because the peptides did not appear to kill the bacteria by interactingwith DNA, we investigated whether they affect the bacterial membraneusing transmission electron microscopy (TEM). A. baumannii strain #1791was treated with buffer (control) or 300 μg/mL P307SQ-8C for 5 minutesor 2 hours. Comparing the TEM images of the samples reveals disruptionof inner membrane and changes in intracellular density (FIGS. 19A, B andC). In addition, we found that the resistant bacteria at pH 7.5 (FIG. 3)were sensitive to P307 at pH 8.8, including E. coli and K. pneumoniae(FIG. 19D). Because the charges on the peptide do not vary as pH changesfrom 7.5 to 8.8, we reasoned that the changes occur on the bacterialmembrane. At higher pH, the bacterial membrane becomes more negativelycharged, allowing the positively charged peptides to establish strongerionic interactions.

Example 19

Without wishes to be bound by theory, we hypothesize the followingmechanism of action: P307SQ-8C interacts with the bacterial membrane togain entry into the cell, and in the process, disrupts the cytoplasmicmembrane. Membrane permeabilization is more effective when the peptideis dimerized. The disruption induces the production of reactive oxygenspecies such as hydroxyl radicals, which disturbs the intracellularcontent. To investigate this hypothesis, we determined membranedisruption using SYTOX® Green uptake assay.

Overnight cultures of bacteria were washed in 50 mM Tris-HCl pH 7.5, andresuspended to an OD₆₀₀ of 0.3 (about 10⁷ cfu/ml). Benzonase® nuclease(25 U/ml)(Novagen) and SYTOX® Green (1 μM) (Invitrogen) was added to thebacterial cells, and incubated for 15 minutes at room temperature in thedark. Peptides were added (50 μg/ml; 14.7 μM P307 and 11.6 μM P307SQ-8C,and melittin (14.7 μM) (Sigma) was used as a control. Relativefluorescence units (RFU) were measured in a SpectraMax Plus reader(Molecular Devices) at room temperature (ex: 485 nm, em: 520 nm) for 2hours. The reactions were carried out twice in duplicate andrepresentative data are shown as mean±standard deviation.

Both peptides permeabilize the membranes of sensitive bacteria, givingrise to an increase in fluorescent signals of SYTOX® Green dye as itbinds to intracellular DNA (FIG. 20). Hydroxyl radical formation wasinvestigated by treating the bacteria with P307 and P307SQ-8C in thepresence of hydroxyl radical scavenger, thiourea. Polymyxin B wasincluded as a control since it has been reported that its bactericidalactivity partially relies on hydroxyl radical death pathway. Thiourea(300 mM) inhibits the activity of P307 and P307SQ-8C completely (FIG.21A). However, it cannot be disregarded that thiourea affects theactivities by other pathways. Therefore, bactericidal activities werealso compared under aerobic and anaerobic conditions. Since A. baumanniiis a strictly aerobic bacteria, E. coli was used for the bactericidalassay with 50 mM Tris-HCl, pH 8.8. Both peptide activities werecompletely inhibited by anaerobic condition (FIG. 21B). Although wecannot rule out other possibilities such as effect on oxygen-dependenttransport mechanism, the current results support our hypothesis ofhydroxyl radical formation.

Example 20

We investigated the in vivo activity of P307SQ-8C using mouse skin modelbecause skin infection is a common route of disease by A. baumannii. Thebacks of 40 female CD-1 mice (6 to 8 weeks of age; Charles RiverLaboratories) were shaved with an electric razor. Nair™ (Hair removerlotion for body and legs, aloe and lanolin) was applied to the shavedareas to remove any remaining hair. The areas were then disinfected withalcohol wipes, and skin abrasion was induced by tape-stripping. An areaof ˜1 cm² of the tape striped skin was then marked and infected with 10μL of about 10⁸ cfu/mL A. baumannii strain no. 1791. The bacteria wereallowed to colonize for 16-18 hours, after which the infected area waseither left untreated or treated with 200 μg of P307SQ-8C or 2 μg ofpolymyxin B for 2 hours. To harvest the remaining bacteria on the skin,the mice were sacrificed and the infected skin was processed in 500 μLPBS for 1 minute in a Stomacher® 80 Biomaster using a microbag (SewardLtd., UK). The solution was serially diluted and plated on LB agarcontaining 4 μg/mL levofloxacin and 12 μg/mL ampicillin for selection.The resulting cfu/mL from each animal is shown as a point and thehorizontal bars represent the means. Both treatments reduce thebacterial load significantly (p-value=0.0023, ordinary one-way ANOVA)(FIG. 22).

What is claimed is:
 1. A polypeptide, wherein the polypeptide comprisesan amino acid sequence having the SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQID NO:20, or SEQ ID NO:21, and the polypeptide is conjugated to anantimicrobial peptide having the amino acid sequence SQSRESQC (SEQ IDNO:44) to yield a conjugated polypeptide and the conjugated polypeptidehas antibacterial activity.
 2. The conjugated polypeptide according toclaim 1, wherein the C-terminus of the polypeptide is conjugated to theantimicrobial peptide.
 3. The conjugated polypeptide according claim 1,wherein the N-terminus of the polypeptide is conjugated to theantimicrobial peptide.
 4. The conjugated polypeptide according to claim1, wherein the conjugated polypeptide has antibacterial activity againsta gram negative bacterium.
 5. The conjugated polypeptide according toclaim 1, wherein the C-terminus of the polypeptide is conjugated to theantimicrobial peptide or the N-terminus of the polypeptide is conjugatedto the antimicrobial peptide.
 6. The conjugated polypeptide according toclaim 5, wherein the conjugated polypeptide has antibacterial activityagainst a gram negative bacterium.
 7. A polypeptide, wherein thepolypeptide comprises an amino acid sequence having the SEQ ID NO:1 orSEQ ID NO:21, or a fragment of the polypeptide consisting of the aminoacid sequence of SEQ ID NO:43 (P307) and the polypeptide or polypeptidefragment is conjugated to an antimicrobial peptide having the amino acidsequence SQSRESQC (SEQ ID NO:44) to yield a conjugated polypeptide andthe conjugated polypeptide has antibacterial activity.
 8. The conjugatedpolypeptide according to claim 7, wherein the C-terminus of theconjugated polypeptide is conjugated to the antimicrobial peptide. 9.The conjugated polypeptide according claim 7, wherein the N-terminus ofthe conjugated polypeptide is conjugated to the antimicrobial peptide.10. The conjugated polypeptide according to claim 7, wherein theconjugated polypeptide has antibacterial activity against a gramnegative bacterium.
 11. The conjugated polypeptide according to claim 7,wherein the C-terminus of the polypeptide or fragment is conjugated tothe antimicrobial peptide or the N-terminus of the polypeptide orfragment is conjugated to the antimicrobial peptide.
 12. The conjugatedpolypeptide according to claim 7, wherein the conjugated polypeptide hasantibacterial activity against a gram negative bacterium.
 13. Thepolypeptide fragment of claim 7, wherein the conjugated polypeptide hasthe amino acid sequence of SEQ ID NO:45.